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Abstract:

A biochip for multiplex genetic identification is disclosed. An biochip
for separating and detecting a plurality of DNA fragments includes a set
of inputs and chambers for receiving a sample matrix of genetic material
and reagents needed to conduct a polymerase chain reaction amplification
of the genetic material. The biochip also includes a plurality of
separation and detection chambers that physically separate different DNA
fragments that have been marked with labels that emit similar colors,
thereby enabling the independent detection of the different DNA
fragments.

Claims:

1. A biochip for multiplex genetic identification, the biochip
comprising: a sample input port to receive a sample containing a
plurality of DNA fragments; and a plurality of separation and detection
chambers in fluid communication with the sample input port, each of the
plurality of separation and detection chambers being spaced apart from
the other chambers, and each chamber containing at least one set of DNA
probes immobilized therein, the set of DNA probes in each chamber
differing from the sets of DNA probes in the other chambers.

2. The biochip of claim 1, further comprising a wash buffer input port to
receive a wash buffer, the wash buffer input port in fluid communication
with the plurality of separation and detection chambers.

3. The biochip of claim 1, further comprising an amplification chamber to
amplify the plurality of DNA fragments, the amplification chamber in
fluid communication with the sample input port and the plurality of
separation and detection chambers.

4. The biochip of claim 3, further comprising: an elution buffer input
port to receive an elution buffer; and a sample preparation chamber in
fluid communication with the elution buffer input port, the sample input
port, and the amplification chamber.

5. The biochip of claim 3, further comprising: a sample preparation wash
buffer input port to receive a sample preparation wash buffer; and a
sample preparation chamber in fluid communication with the sample
preparation wash buffer input port, the sample input port, and the
amplification chamber.

6. The biochip of claim 3, further comprising: a post-amplification
buffer input port to receive a post-amplification buffer; and a
post-amplification vent chamber in fluid communication with the
post-amplification buffer input port and the amplification chamber.

7. The biochip of claim 3, further comprising a post-amplification vent
chamber in fluid communication with the amplification chamber and the
plurality of separation and detection chambers.

8. The biochip of claim 3, further comprising a waste output port in
fluid communication with the plurality of separation and detection
chambers.

9. The biochip of claim 1, further comprising a plurality of vent
chambers, each vent chamber in fluid communication with its corresponding
chamber of the plurality of separation and detection chambers.

10. The biochip of claim 1, further comprising at least one background
reference chamber not in fluid communication with the sample input port
and the plurality of separation and detection chambers, wherein the at
least one background reference chamber and at least one of the plurality
separation and detection chambers are composed of a same material.

11. The biochip of claim 10, wherein each of the at least one background
reference chamber contains at least one DNA probe identical to the at
least one DNA probe of its corresponding separation and detection
chamber.

12. The biochip of claim 1, wherein the plurality of separation and
detection chambers are connected in parallel.

13. The biochip of claim 1, wherein the plurality of separation and
detection chambers are connected in series.

14. The biochip of claim 1, further comprising at least one flow gate to
control fluid flow between the sample input port and at least one of the
plurality of separation and detection chambers.

15. The biochip of claim 14, wherein the at least one flow gate has an
inlet channel and an outlet channel, the at least one flow gate
inhibiting fluid flow from the inlet channel to the outlet channel with
an absence of fluid pressure and permitting fluid flow from the inlet
channel to the outlet channel with fluid pressure.

16. The biochip of claim 14, wherein the at least one flow gate has an
inlet channel and an outlet channel, the at least one flow gate
inhibiting fluid flow from the inlet channel to the outlet channel with
pneumatic pressure and permitting fluid flow from the inlet channel to
the outlet channel with an absence of pneumatic pressure.

17. The biochip of claim 1, further comprising a plurality of transition
channels in fluid communication, wherein each of the plurality of
transition channel has an increasing cross-sectional area in a direction
to its corresponding separation and detection chamber.

18. The biochip of claim 1, further comprising a waste output port in
fluid communication with the plurality of separation and detection
chambers.

19. A flow gate, comprising: a first port and a second port separated by
a junction, each port being a small chamber at an end of a channel; and a
thin flexible material adhered across the first and second ports, wherein
the flow gate has an open configuration and a closed configuration,
wherein pressure applied to the thin flexible material changes between
the open configuration and the closed configuration, the open
configuration having space between the thin flexible membrane and the
junction and permitting fluid flow between the two channels through the
ports, and the closed configuration having the thin flexible membrane in
contact with the junction and inhibiting fluid flow between the two
channels.

21. The flow gate of claim 19, wherein the pressure applied to the thin
flexible material is pneumatic pressure.

22. The flow gate of claim 19, further comprising at least one layer of
material disposed above the ports.

23. The flow gate of claim 22, wherein the flow gate is in the open
configuration without the pressure and wherein the pressure changes the
flow gate from the open configuration to the closed configuration.

24. The flow gate of claim 22, wherein the flow gate is in the closed
configuration without the pressure and wherein the pressure changes the
flow gate from the closed configuration to the open configuration.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application
No. 61/678,364, entitled "Functionally Integrated Device for Multiplex
Genetic Identification," filed Aug. 1, 2012, the contents of which are
incorporated by reference herein.

[0004] The invention generally relates to methods and systems for
multiplex DNA analysis in an integrated biochip, and, more specifically,
to methods and systems that enable multiple target DNA fragments in a
sample to be detected while reducing interference between the detected
targets.

[0005] 2. Description of Related Art

[0006] Integrated biochips, also referred to as microfluidics or
lab-on-a-chip, have gained increasing attention in recent years
especially in clinical diagnostics since they are amiable for
self-contained, portable, point of care devices. These devices enable
performing several biotechnology tasks in a single device thereby
minimizing the need for a large laboratory setup and skilled laboratory
personal to perform diagnostic and research tests. Some of the popular
applications of biochips include cancer detection, environmental testing,
forensics, pathogen identification, gene-expression, SNP detection, to
name a few.

[0007] To date there have been several demonstration of integrated
biochips, some of which illustrate processing sample for cell lysis, DNA
extraction, PCR (polymerase chain reaction) or isothermal amplification
and detection. We use the genetic term "amplification" to mean PCR and
isothermal amplifications. These terms are interchangeable unless
specified otherwise. For example, the integration of sample preparation
and amplification to detection strategies such as capillary
electrophoresis, mass spectroscopy, qPCR, and microarray have been widely
demonstrated. However, it is known that the final DNA fragment detection
method determines the limitations on the number and size of the DNA
fragments that can be amplified by PCR or isothermal amplification.

[0008] Typically, one amplified DNA fragment can correspond to one
organism/disease identification. Hence, the ability to amplify several
DNA fragments and uniquely detect each of these fragments provides a
better platform for identification from a single experiment, both for
research and clinical diagnostics. In this regard, in recent years
multiplex PCR and isothermal amplification reactions have gained
attention since it is capable of generating several DNA fragments to
identify many organisms/diseases from a single reaction. Nevertheless, it
is the DNA detection strategy employed post multiplex amplification that
determines the number (or size) of the DNA fragments that can be uniquely
detected.

[0009] Capillary electrophoresis (CE) and mass spectroscopy are popular
approaches for detecting several DNA fragments generated by multiplex
PCR/isothermal amplification. However, both these methods impose the
limitation that the amplified DNA fragments must be of unique sizes and
the sizes of the DNA fragments should be larger than the separation
resolution of the instrument/system. Furthermore, both capillary
electrophoresis and mass spectroscopy suffer from an inability to
discriminate non-specific amplification if those non-specific DNA
fragments are similar in size to any of the other DNA fragments generated
by multiplex PCR/isothermal amplification. Also, since capillary or
microfluidic electrophoresis in biochips utilize micro fluidic path for
DNA migration, they require expensive excitation and emission optics
(e.g., lasers, CCD, photomultipliers) to sensitively detect low
concentration fluorescence in biochips. Nevertheless, the proven approach
of CE for DNA fragment analysis has rendered this method for the
widespread use in integrated biochips for over a decade.

[0010] In contrast, traditional qPCR using the TaqMan probe method has
better specificity but is limited to the detection of DNA fragments that
can be labeled by uniquely colored fluorophore, determined by the
instrument color detection capability (e.g., known systems are believed
to be limited to nine colors). Hence, integrated biochips that utilize
the qPCR method are limited to scanning and identifying fewer than nine
DNA fragments, if the capability of a nine color qPCR system is employed.
However, qPCR systems typically only demand low cost excitation and
emission optics (e.g. LED, flash lamps, and photodiodes) and this enables
low-cost instrumentation for this method.

[0011] Another approach that increases detection capability for multiplex
PCR/isothermal amplification is the use of microarray technology coupled
with PCR/isothermal amplification. In such a method, a biochip that
performs PCR or isothermal amplification is then coupled with a
microarray method downstream. The feasibility to spot several hundred
detection regions in the microarray technology makes it appealing for
multiplex PCR/isothermal amplification. However, similar to most high
sensitivity electrophoresis systems, microarray detection also requires
the use of a high power spot-excitation source for the fluorophores hence
demanding the use of expensive fluorescence excitation and optical
detection components (e.g. lasers, CCD, photomultipliers).

[0013] Meanwhile, the DNA fragment detection strategy employed in the
integrated biochip, methods, and systems described herein enable a much
higher degree of multiplexing as compared to traditional qPCR, while only
requiring similar inexpensive optics conventionally used in qPCR. The
increased fragment detection capability enables the disclosed techniques
to identify more organisms/diseases from a single test as compared to the
popular and conventional qPCR technology. Thus, embodiments of the
invention enable the use of the relatively low-cost instrumentation of
qPCR optics while increasing the fragment detection capability by several
folds.

[0014] Furthermore, certain embodiments of the invention include universal
fit Luer taper-type connectors integrated into the surface of the biochip
for quick connection while maintaining an air/water-tight seal of reagent
pre-filled syringes/cartridges to the biochip.

BRIEF SUMMARY OF THE INVENTION

[0015] Embodiments of the invention include a functionally integrated
biochip for multiplex genetic identification. The integrated biochip
includes a plurality of separation and detection chambers for receiving a
sample material that contains a plurality of sets of DNA fragments of
different types. Each of the plurality of separation and detection
chambers is spaced apart from each of the other chambers, and each of the
separation and detection chambers has a set of DNA probe disposed
therein. The set of DNA probes disposed in each of the separation and
detection chambers differ in type from those disposed in the other
chambers.

[0016] In some embodiments, the biochip also includes inputs and chambers
for receiving a sample matrix of genetic material and reagents needed to
conduct a polymerase chain reaction or isothermal amplification of the
genetic material to supply the plurality of collections of DNA fragments.

[0017] In other embodiments, to minimize operator intervention and to
realize a completely closed biochip system, an integrated reagent
cartridge coupled to the biochip which stores all required buffers and
waste liquid is provided.

[0018] In some embodiments, a biochip for multiplex genetic identification
is provided. The biochip includes a sample input port to receive a sample
containing a plurality of DNA fragments and a plurality of separation and
detection chambers in fluid communication with the sample input port.
Each of the plurality of separation and detection chambers is spaced
apart from the other chambers, and each chamber contains at least one set
of DNA probes immobilized therein. The set of DNA probes in each chamber
differs from the sets of DNA probes in the other chambers.

[0019] In other embodiments, the biochip includes a wash buffer input port
to receive a wash buffer. The wash buffer input port is in fluid
communication with the plurality of separation and detection chambers.

[0020] In some embodiments, the biochip also includes an amplification
chamber to amplify the plurality of DNA fragments. The amplification
chamber is in fluid communication with the sample input port and the
plurality of separation and detection chambers. In some embodiments, the
biochip includes an elution buffer input port to receive an elution
buffer and a sample preparation chamber in fluid communication with the
elution buffer input port, the sample input port, and the amplification
chamber. In other embodiments, the biochip includes a sample preparation
wash buffer input port to receive a sample preparation wash buffer and a
sample preparation chamber in fluid communication with the sample
preparation wash buffer input port, the sample input port, and the
amplification chamber. In other embodiments, the biochip includes a
post-amplification buffer input port to receive a post-amplification
buffer and a post-amplification vent chamber in fluid communication with
the post-amplification buffer input port and the amplification chamber.
In some embodiments, the biochip includes a post-amplification vent
chamber in fluid communication with the amplification chamber and the
plurality of separation and detection chambers. In other embodiments, the
biochip also includes a waste output port in fluid communication with the
plurality of separation and detection chambers.

[0021] In some embodiments, the biochip includes a plurality of vent
chambers. Each vent chamber is in fluid communication with its
corresponding chamber of the plurality of separation and detection
chambers.

[0022] In other embodiments, the biochip includes at least one background
reference chamber not in fluid communication with the sample input port
and the plurality of separation and detection chambers. The at least one
background reference chamber and at least one of the plurality separation
and detection chambers are composed of a same material. In some
embodiments, each of the at least one background reference chamber
contains at least one DNA probe identical to the at least one DNA probe
of its corresponding separation and detection chamber.

[0023] In some embodiments, the plurality of separation and detection
chambers are connected in parallel. In alternative embodiments, the
plurality of separation and detection chambers are connected in series.

[0024] In other embodiments, the biochip includes at least one flow gate
to control fluid flow between the sample input port and at least one of
the plurality of separation and detection chambers.

[0025] In some embodiments, the at least one flow gate has an inlet
channel and an outlet channel. The at least one flow gate inhibits fluid
flow from the inlet channel to the outlet channel with an absence of
fluid pressure and permits fluid flow from the inlet channel to the
outlet channel with fluid pressure.

[0026] In alternative embodiments, the at least one flow gate has an inlet
channel and an outlet channel. The at least one flow gate inhibits fluid
flow from the inlet channel to the outlet channel with pneumatic pressure
and permits fluid flow from the inlet channel to the outlet channel with
an absence of pneumatic pressure.

[0027] In some embodiments, the biochip includes a plurality of transition
channels in fluid communication. Each of the plurality of transition
channel has an increasing cross-sectional area in a direction to its
corresponding separation and detection chamber.

[0028] In other embodiments, the biochip includes a waste output port in
fluid communication with the plurality of separation and detection
chambers.

[0029] In some embodiments, a flow gate is provided. The flow gate
includes a first port and a second port separated by a junction, each
port being a small chamber at an end of a channel. The flow gate also
includes a thin flexible material adhered across the first and second
ports. The flow gate has an open configuration and a closed
configuration, wherein pressure applied to the thin flexible material
changes between the open configuration and the closed configuration. The
open configuration has space between the thin flexible membrane and the
junction and permits fluid flow between the two channels through the
ports, and the closed configuration has the thin flexible membrane in
contact with the junction and inhibits fluid flow between the two
channels.

[0030] In some embodiments, the thin flexible material is plastic.

[0031] In some embodiments, the pressure applied to the thin flexible
material is pneumatic pressure.

[0032] In some embodiments, the flow gate includes at least one layer of
material disposed above the ports.

[0033] In some embodiments, the flow gate is in the open configuration
without the pressure, and the pressure changes the flow gate from the
open configuration to the closed configuration.

[0034] In other embodiments, the flow gate is in the closed configuration
without the pressure, and the pressure changes the flow gate from the
closed configuration to the open configuration.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0035] FIG. 1 illustrates a biochip that includes separation and detection
chambers and a serial flow path according to an embodiment of the
invention.

[0036]FIG. 2 illustrates a single type of probe immobilized within a
separation and detection chamber according to an embodiment of the
invention.

[0037]FIG. 3 illustrates more than one type of probe immobilized within a
separation and detection chamber according to an embodiment of the
invention.

[0038]FIG. 4 is a flowchart illustrating a series of steps for using the
biochip shown in FIG. 1 according to an embodiment of the invention.

[0039] FIG. 5 illustrates a biochip that includes separation and detection
chambers and a parallel flow path according to an embodiment of the
invention.

[0040] FIG. 6 is a flowchart illustrating a series of steps for using the
biochip shown in FIG. 5 according to an embodiment of the invention.

[0041] FIG. 7 illustrates an integrated biochip that includes sample
preparation, target separation, and target detection features according
to an embodiment of the invention.

[0042] FIG. 8 is a flowchart illustrating a series of steps for using the
biochip shown in FIG. 7 according to an embodiment of the invention.

[0043] FIG. 9 illustrates an integrated biochip that includes universal
LUER-LOK® connections integrated into the biochip according to an
embodiment of the invention.

[0044] FIG. 10A illustrates a flow gate of an integrated biochip according
to an embodiment of the invention.

[0045] FIG. 10B illustrates an open configuration of the flow gate of FIG.
10A according to an embodiment of the invention.

[0046] FIG. 10C illustrates a closed configuration of the flow gate of
FIG. 10A according to an embodiment of the invention.

[0047] FIG. 11A illustrates an angled view of an integrated biochip with
an integrated cartridge according to an embodiment of the invention.

[0048] FIG. 11B illustrates a bottom view of the integrated biochip of
FIG. 10A according to an embodiment of the invention.

[0049] FIG. 12 illustrates an integrated biochip configured to be
connected to an integrated cartridge according to an embodiment of the
invention.

[0050] FIG. 13 illustrates an integrated cartridge configured to be
connected to an integrated biochip according to an embodiment of the
invention.

[0051] FIG. 14A illustrates a buffer cartridge assembly with a
self-sealing rubber gasket according to an embodiment of the invention.

[0052] FIG. 14B illustrates inserting a sample to a buffer cartridge
according to an embodiment of the invention.

[0053] FIG. 15A illustrates a cross-sectional side view of a cartridge,
membrane and biochip stack according to an embodiment of the invention.

[0054] FIG. 15B illustrates a cross-sectional view (B-B) of the cartridge
of FIG. 15A according to an embodiment of the invention.

[0055] FIG. 15C illustrates a cross-sectional view (C-C) of the membrane
of FIG. 15A according to an embodiment of the invention.

[0056] FIG. 15D illustrates a cross-sectional view (DE-DE) of the biochip
of FIG. 15A according to an embodiment of the invention.

[0057] FIG. 15E illustrates a cross-sectional view (DE-DE) of the biochip
of FIG. 15A according to an embodiment of the invention.

[0058] FIG. 16A illustrates a cross-sectional side view of the stack of
FIG. 15A with an open membrane slit according to an embodiment of the
invention.

[0059] FIG. 16B illustrates a cross-sectional view (B-B) of the membrane
of FIG. 16A with an open slit according to an embodiment of the
invention.

[0060] FIG. 17A illustrates a cross-sectional side view of the stack of
FIG. 15A with a closed membrane slit according to an embodiment of the
invention.

[0061] FIG. 17B illustrates a cross-sectional view (B-B) of the membrane
of FIG. 17A with a closed slit according to an embodiment of the
invention.

[0062] FIG. 18 illustrates a testing platform according to an embodiment
of the invention.

[0064] FIG. 19B lists examples of common quencher sequences with
dye-quenching moieties according to an embodiment of the invention.

[0065]FIG. 20 illustrates increases in relative fluorescence for four
tested targets according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0066] Embodiments of the invention detect, by fluorescence, multiple DNA
targets of similar or different size within a biochip by separating the
DNA fragments into designated chambers in the biochip. Herein, this DNA
analysis method is termed "space separation and detection". Using the
space separation and detection method offers several key advantages that
are not offered by routinely used DNA detection methods such as
electrophoresis, mass spectroscopy, qPCR, etc. Embodiments of the
invention include an integrated biochip that is capable of accepting a
raw sample (such as blood, saliva, urine, swabs) to process via cell
lysis, DNA extraction, DNA purification, PCR or isothermal amplification
of purified DNA, post-amplification fragment preparation, and, finally,
space separation in designated chambers. All processes are performed on a
single use disposable integrated biochip capable of "sample-to-results"
for research and clinical diagnostics.

[0067] FIG. 1 illustrates a biochip 100 that includes separation and
detection chambers 105, 110 and a serial flow path 115 according to an
embodiment of the invention. The biochip 100 also includes a sample input
port 120, a wash buffer input port 125, and a waste output port 130.
Channels 135 connect the various features of the biochip 100. Flow gates
140 and vent chambers 145 control fluid flow through the channels 135.
The biochip 100 also includes a reference chamber 150, discussed in more
detail below.

[0068] Biochip 100 is configured to receive a sample that contains a
collection of DNA fragments, sequentially capture one or more sets of DNA
fragments in the separation and detection chambers 105, 110 (and others),
and enable detection of the various fragments. The sample to be analyzed
is introduced via the sample input port 120 and flows through one of the
channels 135 to a flow gate 140 that controls access to separation and
detection chamber 105. Upon opening the flow gate to separation and
detection chamber 105, the sample mixture flows into chamber 105 under
pressure applied at sample input port 120. The vent chamber 145
immediately downstream of chamber 105 permits gas present in the channels
upstream of chamber 105, and in chamber 105 itself, to be vented outside
of the biochip. The structure and operation of the flow gates and vent
chambers are described in more detail below.

[0069] Separation and detection chamber 105 is immediately preceded by a
transition channel 155. The transition channel expands the
cross-sectional area of the channel exiting the flow gate to more closely
match the entrance to the chamber 105. The transition channel 155 may be
configured to expand the cross-sectional area in a stepwise manner, as
shown in the figure, or it may be configured to do so in a continuous
manner. Moreover, although only three distinct sections of the transition
channel 155 are shown in the figure, other numbers of sections are within
the scope of the invention.

[0070] In an illustrative implementation, each sequential section of the
transition channel 155 increases the cross-sectional area of the channel
about two to four times relative to the preceding channel. Other ratios
may be employed. By gradually increasing the cross-sectional area of the
channel leading to the separation and detection chamber 105, a sudden
transition from a relatively narrow passage to a relatively large chamber
is avoided. This, in turn, reduces or avoids the occurrence of gas
bubbles forming in the chamber 105. By reducing or eliminating gas
bubbles in chamber 105, the design of the biochip 100 enables detection
errors to be reduced or eliminated, thereby providing relatively higher
quality results than without such features.

[0071] As will be described in more detail in connection with FIG. 2,
certain steps are taken to retain one or more of the DNA fragments in the
separation and detection chamber 105 before passing the sample into a
separation and detection chamber 110. In order to pass the sample mixture
from chamber 105 into chamber 110, the flow gates 140 upstream of chamber
110 are opened, and the sample mixture flows, under pressure from the
sample input port 120, into chamber 110. As described above, a vent
chamber 145 immediately downstream of chamber 110 permits gas in the
channels and the chamber 110 to escape. Also as before, certain steps are
taken to retain one or more of the DNA fragments in the separation and
detection chamber 110 before passing the sample into subsequent
separation and detection chambers following the same basic procedure as
described above.

[0072] After passing the sample mixture into separation and detection
chamber 160 and performing the steps needed to retain one or more of the
DNA fragments of the sample mixture in chamber 160, the flow gate
immediately downstream of the chamber 160 is opened, along with all flow
gates preceding chamber 160, to permit introduction of a wash buffer
fluid through the wash buffer input port 125. The wash buffer fluid
passes through all of the separation and detection chambers and exits via
the waste output port 130. The flow of wash buffer fluid through the
chambers reduces the amount of sample mixture remaining in the separation
and detection chambers with the exception of the DNA fragments that have
been bound within the chambers. Although biochip 100 is shown as having
six separation and detection chambers, more or fewer chambers may be
included and remain within the scope of the invention.

[0073] As mentioned above, certain steps are taken to retain one or more
DNA fragments in the various separation and detection chambers. In an
illustrative implementation of the invention, a set of DNA probes are
immobilized within each separation and detection chamber. As used herein,
a DNA probe is an agent that binds directly to a predefined sequence of
nucleic acids. In general, DNA probes can be labeled or unlabeled, as
described herein. FIG. 2 illustrates a single type of probe immobilized
within a separation and detection chamber 200 according to an embodiment
of the invention. FIG. 2 shows a cross-section of one of the chambers
(e.g., any of chambers 105, 110, or the others shown in FIG. 1). The
chamber 200 has a top surface 205, a bottom surface 210 and sidewalls 215
and 220. Techniques for forming the chamber 200 are discussed in greater
detail below. In general, however, a void is created in a substrate to
form the sidewalls 215, 220.

[0074] In some implementations, the top surface 205 is part of the
substrate into which the chamber 200 is formed. In other implementations,
bottom surface 210 is part of the substrate. In still further
implementations, top surface 205 and bottom surface 210 are formed of a
plastic and/or glass material. Using glass as an illustrative example, a
set of DNA probes 225, all of the same type, are immobilized onto a glass
slides (˜1 mm thick). One glass slide is mated and sealed above the
chamber 200 to form the top surface 205 and another glass slide is mated
and sealed below the chamber 200 to form the bottom surface 210, thereby
creating a visual detection window. Thus, in the embodiment shown in FIG.
2, the binding locations for the DNA probes 225 are located on the "roof"
and "floor" of the chamber. Although not shown, DNA probes can be
immobilized on sidewalls 215, 220 in addition to or in place of those
immobilized on the top and bottom surfaces.

[0075] One of many illustrative techniques for immobilizing the DNA probes
onto the glass slide follows. In some embodiments, desalted 3-prime or
5-prime amine-modified oligonucleotide probes are used. The DNA probe
material is first mixed in an immobilization buffer (e.g., 300 nM sodium
phosphate (pH 8.0), Polysorbate 20 0.005%, and sarkosyl 0.001%) at a
concentration of about 20 μM. A drop of the immobilization buffer
solution that is about the size of the detection chamber (e.g., 3.5 mm)
or several small droplets (e.g., 0.2 mm or smaller) within a space that
is roughly the size of the detection chamber, are made on the glass slide
using a micropipette or other robotic dispensing instruments. The glass
slide is kept in a humidified chamber (e.g., 50% relative humidity) for
few to several minutes (e.g., up to 30 minutes) and then soaked in a
deactivation buffer (e.g., 50 nM ethanolamine in 50 nM sodium borate
solution of pH 9.0) for about 30 minutes to 1 hour. The deactivation
buffer ensures that any region that does not have an immobilized probe(s)
is made relatively chemically inert to reduce the likelihood of DNA
material adhering to those regions during the probe-DNA interaction.
Finally, the glass is rinsed with deionized water for about 1 minute. The
glass slide is then affixed to the substrate above the separation and
detection chamber(s).

[0076] The above technique is merely illustrative, and other known methods
for immobilizing probes may be used. In addition, the process set forth
above can be used to immobilize DNA probes on the sidewalls of the
chambers. The probes can be immobilized to particles (e.g., magnetic
beads of 10 nm to 10 μm) and can be placed inside the separation and
detection chamber(s) instead of the glass coating method described above.
For example, if magnetic particles with immobilized probes were used, a
magnet can be energized above and/or below the chamber to retain the
magnetic particles during fluid flow.

[0077] Referring again to FIG. 1, the biochip 100 also includes a
background reference chamber 150. Chamber 150 is formed in the same
manner and with the same materials as separation and detection chambers
105, 110, and the other chambers. However, chamber 150 is isolated from
the path taken by the sample mixture and, in some embodiments, does not
contain any immobilized DNA probes. Thus, any background fluorescence
caused by the materials that form the top surface, bottom surface, and
sidewalls can be detected. This background fluorescence can then be
subtracted from the fluorescence from the separation and detection
chambers in order to improve accuracy of the test results.

[0078] In other embodiments, one or more DNA probes of the type(s) used in
the separation and detection chambers are immobilized in chamber 150.
Thus, any background fluorescence caused by the DNA probes, in addition
to that contributed by the materials of construction, can also be
detected and subtracted from final test results. Further still, other
implementations more than one background reference chamber is included in
biochip 100. For example, biochip 100 can include one background
reference chamber corresponding to each separation and detection chamber
(105, 110, and the other) present in biochip 100. In such an embodiment,
each of the background reference chambers includes the same type and
concentration of immobilized DNA probes present in the corresponding
separation and detection chamber. Thus, any variability of background
fluorescence due to the different DNA probes can be detected and
accounted for.

[0079] The visual detection window created by this configuration enables
the detection and quantification of fluorescence (for techniques using
fluorescent labels or PCR primers) from the DNA probe/target sequence
pair from either above or below the surface of the biochip. Furthermore,
the use of glass in the structure is advantageous as it is optically
clear, has better binding chemistries for the probes, and has low
auto-fluorescence compared to most plastics. However, it is understood
that the use of plastics in place of glass remains within the scope of
the invention.

[0080]FIG. 3 Illustrates more than one type of probe immobilized within a
separation and detection chamber 300 according to an embodiment of the
invention. Chamber 300 shares similar features to the chamber 200 of FIG.
2. However, chamber 300 has two types of DNA probes immobilized within
the chamber. DNA probes 305 and 310 each bind to different predefined
sequences of nucleic acids. Thus, during the separation phase, described
in more detail below, two different DNA fragment targets are retained in
chamber 300.

[0081]FIG. 4 is a flowchart illustrating a series of steps for using the
biochip shown in FIG. 1 according to an embodiment of the invention.
Initially, a sample (e.g., blood, saliva, etc.,) is gathered and prepared
to create a collection of target DNA fragments using techniques known to
one having ordinary skill in the art. The DNA fragments are then
amplified using PCR or other DNA amplification techniques such as
isothermal amplification (step 405). The particular amplification process
and reagents used are determined in part by the nature of the DNA probes
immobilized in the separation and detection chambers (105, 110, and the
others) as well as the intended process for detecting the presence of the
DNA targets.

[0082] For example, the PCR/isothermal process can produce amplified DNA
fragments that are fluorescently labeled, which, when captured by the
immobilized DNA probes, cause the fluorescence that is ultimately
detected. In other implementations, the DNA probes include a fluorescent
label and the amplified DNA fragments are not labeled. A quencher
compound is included in the sample mixture along with the DNA fragments
that are passed to the separation and detection chambers. When the DNA
target fragments bind to the probes, the fluorescence of those probes is
preserved, while the fluorescence of any DNA probes without bound DNA
targets is quenched. Thus, in such embodiments, the probes cause the
fluorescence that is ultimately detected. A more detailed description of
the various illustrative complementary configurations of DNA probes,
PCR/isothermal amplification processes, and detection techniques that can
be used in biochip 100 are set forth in the incorporated application and
references included in the appendix.

[0083] For illustrative purposes in connection with the process set forth
in FIG. 4, six different types of DNA fragments (fragments types 1-6
herein) are amplified, and the DNA fragments produced by the
PCR/isothermal amplification process (step 405) are fluorescently labeled
with the same fluorescent label (e.g., green dye FAM). Also for the
purpose of this example, each of the six separation and detection
chambers (105, 110, and the others) has a different DNA probe immobilized
therein that are complementary to the six different types of DNA
fragments. After the amplification process, the collection of DNA
fragments and any reagents or buffers that are added after amplification
is complete (collectively called "post-amplification solution") are
introduced to biochip 100 via the sample input port 120 (step 410). As
described in greater detail above, one or more flow gates are opened to
permit the post-amplification solution to pass into the desired features
of the biochip 100.

[0084] Next, the post-amplification solution, containing the collection of
six different types of DNA fragments, is passed to the next available
separation and detection chamber (step 415), which at this point in the
process is the first chamber 105. The contents of the first chamber 105
are then subject to a thermo-chemical process to bind fragments of type 1
to the immobilized DNA probes in the first separation and detection
chamber 105 (step 420). After the binding process is complete, the
post-amplification solution is passed to the next available separation
and detection chamber (repeat step 415), which is now chamber 110. Again,
a thermo-chemical binding process is performed to capture DNA fragments
of type 2, which are complementary to the DNA probes in the second
separation and detection chamber 110, within the second chamber (repeat
step 420).

[0085] Steps 415 and 420 are repeated until the post-amplification
solution has passed into each of the six separation and detection
chambers and each of the six different types of DNA fragments have been
bound to the DNA probes to which they correspond. After the final binding
step (step 420), the remaining post-amplification solution is passed to
waste output port 130 (step 425). The illustrative process next passes a
separation wash buffer, which is introduced via wash buffer input port
125, through all of the separation and detection chambers to waste output
port 130 (step 430). The wash buffer removes unbound DNA fragments from
the separation and detection chambers.

[0086] Finally, the fluorescence of the six separation and detection
chambers 105, 110, and the others (and, optionally, the background
reference chamber 150) are detected and quantified (step 435). Any of the
equipment for and methods of detecting and quantifying the fluorescence
known to one having ordinary skill in the art can be used during this
step. For example, use of fluorometers appropriate for use in
Quantitative PCR (qPCR) techniques are within the scope of the invention.
Because each separation and detection chamber 105, 110, and the others
had a different DNA probe immobilized therein, each chamber captured a
different type of DNA fragment. Thus, embodiments of the invention enable
one to use the same fluorescent label for each of the plurality of
different DNA types in the sample without the problem of being unable to
distinguish between the various DNA types.

[0087] FIG. 5 illustrates a biochip 500 that includes separation and
detection chambers 505 and a serial flow path 515 according to another
embodiment of the invention. The biochip 500 also includes a sample input
port 520 and channels 535 connect the various features of the biochip
500. Flow gates 540 and vent chambers 545 control fluid flow through the
channels 535. The biochip 500 also includes a reference chamber 550,
discussed in more detail above.

[0088] Biochip 500 is similar in many respects to the biochip 100
described in detail above. However, biochip 500 is configured to receive
the post-amplification solution and distribute the solution among six
channels that form parallel flow paths 515 in contrast to the serial flow
path 115 of biochip 100. In this context, "parallel flow" means that the
sample solution passes into a particular separation and detection chamber
505 without having passed through any other separation and detection
chamber 505. In this regard, the channels that form the flow paths need
not be strictly geometrically parallel (as shown in FIG. 5A and FIG. 5B),
however, the paths can be geometrically parallel. In addition, it is
understood that the post-amplification solution need not enter the
channels or the separation and detection chambers 505 at exactly the same
time.

[0089] The sample to be analyzed is introduced via the sample input port
520 and flows through the channels 535 to flow gates 540 that control
access to separation and detection chambers 505. Upon opening the flow
gates to the multiple separation and detection chambers 505, the sample
mixture flows into chambers 505 under pressure applied at sample input
port 520. The vent chambers 545 immediately downstream of chambers 505
permit gas present in the channels upstream of chambers 505, and in the
chambers 505 themselves, to be vented outside of the biochip. The
structure and operation of the flow gates and vent chambers are described
in more detail below.

[0090] Separation and detection chambers 505 include DNA probes therein.
However, unlike the serial embodiment of the biochip 100, it is not
required that the DNA probes be immobilized in the chambers 505 because
the portion of post-amplification solution that enters each of the
chambers 505 does not leave the chamber. Although not required, it is
understood that in some implementations of the parallel flow embodiment
of biochip 500, one or more DNA probes may be immobilized in the chambers
505 using the techniques described above.

[0091] Another difference between the serial flow biochip 100 and the
parallel flow biochip 500 is that the step of binding the desired DNA
fragments of the sample to the probes in the multiple chambers 505 need
not happen in sequence. Rather, all of the separation and detection
chambers 505 can be subjected to a process for binding the desired DNA
fragments to the DNA probes in the same set of steps. In the alternative,
one or more of the chambers 505, but less than all chambers, can be
subjected to a binding process at the same time. For example, a first set
of three chambers can be subjected to a thermo-chemical binding process
during a first set of binding steps, while the remaining three chambers
can be subjected to the same or similar set of binding steps after the
first set of binding steps is completed. Although biochip 500 is shown as
having six separation and detection chambers, more or fewer chambers may
be included and remain within the scope of the invention.

[0092] FIG. 6 is a flowchart illustrating a series of steps for using the
biochip 500 shown in FIG. 5 according to an embodiment of the invention.
As with the process for using biochip 100, a sample is gathered and
prepared to create a collection of target DNA fragments using techniques
known to one having ordinary skill in the art and the DNA fragments are
then amplified using PCR or other DNA amplification techniques (e.g.,
isothermal amplification) (step 605). As before, the particular
PCR/isothermal amplification process and reagents used are determined in
part by the nature of the DNA probes present in the separation and
detection chambers 505 as well as the intended process for detecting the
presence of the DNA targets.

[0093] For illustrative purposes in connection with the process set forth
in FIG. 6, six different types of DNA fragments (fragments types 1-6
herein) are amplified (step 605). However, unlike the process previously
described, the DNA fragments produced by the PCR or isothermal
amplification process (step 605) are not fluorescently labeled. Rather,
the DNA probes present in the chambers 505 are labeled with a fluorescent
label at either the 3-prime end or 5-prime end of the probe. Meanwhile,
the unlabeled ends of the DNA probes have quenchers thereon. The opposing
ends of the DNA probes have a number of bases that are complementary to
each other. Also for the purpose of this example, each of the six
separation and detection chambers 505 has a different type of DNA probe
present therein that is complementary to one of the six different types
of DNA fragments. After the amplification process, the collection of DNA
fragments and any reagents or buffers that are added after amplification
is complete (collectively called "post-amplification solution") are
introduced to biochip 500 via the sample input port 520 (step 610). As
described in greater detail above, one or more flow gates are opened to
permit the post-amplification solution to pass into the desired features
of the biochip 500.

[0094] Next, the post-amplification solution, containing the collection of
six different types of DNA fragments, is passed to the separation and
detection chambers 505 (step 615). The contents of the chambers 505 are
then subject to a thermo-chemical process to bind each type of DNA
fragment to its complementary type of DNA probe (step 620). In this
embodiment, when a DNA fragment binds to its complementary DNA probe, the
fluorescence of the probe is maintained. Any DNA probes in any of the
chambers 505 that are not bound to a complementary DNA fragment will
self-quench via the complementary bases at the 3-prime and 5-primes ends
binding to each other.

[0095] After the binding process is complete, the fluorescence of the six
separation and detection chambers 505 (and, optionally, the background
reference chamber 550) are detected and quantified (step 625). As
mentioned above, any of the equipment for and methods of detecting and
quantifying the fluorescence known to one having ordinary skill in the
art can be used during this step. Because each of the separation and
detection chambers 505 had a different DNA probe therein, each chamber
detects a different type of DNA fragment.

[0096] FIG. 7 illustrates an integrated biochip 700 that includes sample
preparation, target separation, and target detection features according
to an embodiment of the invention. The biochip 700 includes a serial flow
path 715 leading through separation and detection chambers 705 that is
similar to that of the biochip 100. However, other embodiments include a
parallel flow path such as the one described above. The biochip 700
includes a wash buffer input port 725, a waste output port 730,
connecting channels 735, flow gates 740, vent chambers 745, and a
reference chamber 750. These features have similar characteristics and
operate in a similar manner to the corresponding features described in
connection with the biochip 100 and biochip 500.

[0097] Biochip 700 is similar in many respects to the biochip 100
described in detail above. However, biochip 700 has additional features
that enable sample preparation and PCR/isothermal amplification to be
performed on the biochip 700. Thus, the biochip 700 also includes a
sample input port 755, a sample preparation wash buffer input port 760,
an elution buffer input port 765, and a sample preparation chamber 770.
As described in more detail below, these additional features are used to
prepare a sample material for PCR/isothermal amplification. The biochip
700 further includes an amplification chamber 775, a post-amplification
buffer input port 780, and a post-amplification vent chamber 785. These
later features enable PCR/isothermal amplification to be performed on the
biochip 700. Other embodiments of the biochip includes some, but not all,
of the components listed above.

[0098] The sample input port 755 permits a sample matrix (e.g., blood,
saliva, urine, swab material) to be introduced into the sample
preparation chamber 770. Similarly, the sample preparation wash buffer
input port 760 permits a sample preparation wash buffer to be introduced
into the sample preparation chamber 770, and the elution buffer input
port 765 permits an elution buffer to added to the mixture in the sample
preparation chamber 770. After the steps needed to liberate the target
DNA fragments are performed on the sample matrix and reagents in the
sample preparation chamber 770, the mixture is introduced into the
amplification chamber 775.

[0099] After PCR thermocycling is conducted, a post-amplification buffer
is introduced to the amplification chamber 775 via the post-amplification
buffer input port 780, and the combined solution is passed to the
post-amplification vent chamber 785. The post-amplification vent chamber
is similar to other vents of the biochips described herein in that it
permits gases in the upstream channels as well as the chamber itself to
escape while retaining the sample solution. Fluid flow between the sample
preparation and PCR amplification features is achieved via the same
techniques set forth in more detail above. Generally, flow gates 740
control the flow of fluid between the various features, and vent chambers
745 permit gas to be purged from the chambers and the connecting
channels.

[0100] The sample to be analyzed is then introduced to the separation and
detection chambers 705 in a manner similar to the one set forth in
connection with the biochip 100. As mentioned above, alternative
embodiments of the biochip 700 include a parallel flow path. In such
embodiments, the post-amplification solution generated by the "front-end"
of biochip 700 passes into the separation and detection chambers in a
manner similar to the one provided above in connection with the biochip
500.

[0101] FIG. 8 is a flowchart illustrating a series of steps for using the
biochip 700 shown in FIG. 7 according to an embodiment of the invention.
Initially, a sample matrix is collected (e.g., a patient's blood) and
combined with a lysis buffer and magnetic particles (step 805). The lysis
buffer gains access to the DNA material inside the blood cells, and the
DNA material binds to the magnetic particles under certain conditions.
Next, a magnet is engaged above the sample preparation chamber 770, and
the sample mixture created in step 805 is passed into the sample
preparation chamber 770 via sample input port 755 (step 810). A sample
preparation wash buffer is introduced to the sample preparation chamber
770 via the sample preparation wash buffer input port 760 and passed
through the chamber 770 to the waste output port 730 (step 815). This
wash step removes unwanted material and contaminants (e.g., cellular
debris) from the sample mixture. However, the desired DNA material
remains in the sample preparation chamber 770 because the material is
bound to the magnetic particles, which are held within the sample
preparation chamber 770 via the magnet engaged above.

[0102] Next, an elution buffer is added to sample preparation chamber 770
via the elution buffer input port 765 (step 820). The sample mixture and
elution buffer are incubated in the sample preparation chamber 770 at the
appropriate conditions (e.g., 50 deg C. for 3 minutes) (step 825), which
would be known to one having ordinary skill in the art. During the
incubation step, the elution buffer causes the DNA material to be
liberated from the magnetic particles. The washed and eluted sample
mixture is then passed into the amplification chamber 775 (step 830),
leaving behind the magnetic particles that are still held in the sample
preparation chamber 770 by the magnet. The needed amplification reagents
can be included in the elution buffer or present in the amplification
chamber 775 (or channels leading to the amplification chamber) in
lyophilized form. In embodiments employing reverse-transcriptase-PCR
(RT-PCR), the RT-PCR reagents are included in the elution buffer and the
RT reaction is performed in the sample preparation chamber 770.
Meanwhile, the amplification reagents are present in the amplification
chamber 775 (or channels leading to the amplification chamber) in
lyophilized form. The sample is then subjected to PCR thermocycling or
isothermal amplification (830) to amplify the desired target DNA
fragments.

[0103] After completion of the amplification reaction by thermocycling in
case of PCR amplification or by constant temperature in case of
isothermal amplification, the amplified products are passed to the
post-amplification vent chamber 785 and a post-amplification buffer is
introduced via the post-amplification buffer input port 780 into the
post-amplification vent chamber 785 (step 835). The amplified products
and post-amplification buffer are mixed in the post-amplification vent
chamber 785 to form the "post-amplification solution". At this point, the
post-amplification solution is passed into the separation and detection
chambers 705 in a serial flow fashion following steps 415-430 of FIG. 4
(step 840). Finally, the fluorescence of the six separation and detection
chambers 705 (and, optionally, the background reference chamber 750) are
detected and quantified. Any of the equipment for and methods of
detecting and quantifying the fluorescence known to one having ordinary
skill in the art can be used during this step.

[0104] FIG. 9 illustrates an integrated biochip 900 that includes
universal LUER-LOK® connections 905 integrated into the biochip
according to an embodiment of the invention. In other embodiments, an
alternate Luer taper-type connection is used (e.g., a LUER-SLIP®).
The LUER-LOK® connectors provide for quick connection between the
biochip and the source of materials for use in the biochip while
maintaining an air/water-tight seal. Alternatively, a buffer cartridge
integrated onto the biochip may be used instead of syringes and
LUER-LOK® as shown in FIGS. 14A and 14B.

[0105] Certain implementations of the various embodiments of biochips
described above include a filter (<50 μm) in or connected to the
biochip's waste output port. The filter in the waste output port prevents
backflow of material from the waste receptacle (e.g., a syringe) to the
biochip when liquid is present in the waste receptacle. Alternatively, a
check valve is in or fitted to the waste output port to serve the same
purpose of backflow prevention achieved using a filter.

[0106] The various implementations of the biochips described herein can be
fabricated from a variety of materials including glass, silicone, soft
polymers (e.g., PDMS), and plastics. Plastic polymers such as Poly(methyl
methacrylate) (PMMA), polypropylene, polycarbonate, polyimide, cyclic
olefin copolymers (COC), as well as glass are generally preferred, as
those materials have good biocompatibility and are stable at high
temperature (>80 C) utilized in bioprocesses. Meanwhile, soft polymers
are less desirable as they generally exhibit gas and water permeability,
especially at elevated temperatures.

[0107] In certain embodiments, the features of the biochips (e.g.,
channels, wells, chambers, etc.) are fabricated in the underside of a
plastic substrate via hot embossing, injection molding, laser machining,
or micro-machining process. It is understood that the foregoing list is
merely illustrative, and other techniques for forming the biochip
features are within the scope of the invention. The plastic substrate
with the features is then bonded to a plastic film (of, e.g., ˜150
μm thickness) onto the underside to create enclosed channels,
chambers, and other features in the biochip. For example, COC can be used
as the substrate and thin film in an embodiment of the biochip. After
forming the features in the substrate, the thin film and substrate are
thermally bound using heat and pressure (e.g., 135° C. at 200 PSI
for about 1 minute). In general, the temperature used is close to the
glass-transition temperature of the plastic material used. Thus, other
temperatures are within the scope of the invention, depending on the
materials of construction. In addition, other pressures and times, e.g.,
ranging from 20 PSI to 1000 PSI and a few seconds to several minutes,
respectively, may be used. A device, such as a Model 4398 laboratory
press from Carver, Inc. may be used to achieve these conditions.

[0108] Another illustrative technique uses heat, pressure, and a solvent
to achieve bonding between the substrate and the thin film. In such a
process, cyclohexane is applied to the surface of the substrate and/or
thin film. The two components are then brought together under about 100
PSI at roughly 70° C. for about 2 minutes. This process produces a
biochip which can withstand internal pressures of at least 100 PSI
without delaminating.

[0109] As shown in FIG. 7, amplification chamber 775 exhibits a meander.
One advantage of this feature is that it reduces or eliminates the
tendency of the plastic film to deflect inward into the channel that it
forms. By reducing or eliminating this deflection, a relatively flatter
surface is available for contacting the heating elements used during the
PCR thermocycling or isothermal amplification. This provides better
temperature control, increased efficiency, and improved performance. In
addition, the meander provides for relatively better mixing than a
chamber with straight channels.

[0110] As set forth above, fluid flow control between the chambers,
channels, reservoirs, wells, and other features is controlled in part by
flow gates (e.g., flow gates 140 of FIG. 1). These flow gates can be any
of a number of valves, flow control features, and the like suitable for
use in microfluidic service known to one having ordinary skill in the
art. In one illustrative implementation, the small chambers at the end of
the channels that form the gate function are about 500 μm in diameter;
larger and smaller diameters may also be used.

[0111] FIG. 10A illustrates a flow gate mechanism in some embodiments. In
these embodiments, a flow gate includes a flexible membrane 1042 and an
adhesive 1044 on one side of the membrane 1042. The flexible membrane
1042 is affixed to the area circumscribing the openings of channels 1001
and 1002, and the adhesive 1044 initially seals the area between the two
disconnected channels.

[0112] Without the seal, fluids in the cartridge may leak or flow into
other areas of the biochip because flow-gate membrane does not seal
airtight when not connected to an instrument. When the adhesive 1044 is
applied, it seals the channel leading to the reservoir (upstream) in the
cartridge. However, upon the first use of the biochip on the instrument,
the fluid pressure will delaminate the adhesive between 1001 and 1002,
breaking the adhesive seal. Once the adhesive 1044 is soiled by the fluid
at its first use as shown in FIG. 10B and FIG. 10C, the membrane at that
flow gate needs a pneumatic pressure to actuate the membrane either to
allow fluid flow in FIG. 10B or to prevent fluid flow in FIG. 10C.
Therefore, in some embodiments, the adhesive 1044 replaces a typical
sealing mechanism (e.g. foil seal) for each reservoir of the cartridge to
prevent fluid from moving out of the reservoir and into other areas of
the biochip.

[0113] FIG. 10B illustrates an open configuration of the flow gate.
Applying pressure 1077B to an end of a channel causes fluid pressure to
apply upward pressure to the flow gate membrane 1042. For example, air
pressure can be applied from the buffer cartridge 1095 to channel 1001 to
the membrane 1042. As a result, the flexible flow gate membrane bends and
allows the fluid to flow through a next channel. Because the adhesive
1044 circumscribes the area, the fluid is restricted to stay within the
area and flows from one channel to another channel.

[0114] FIG. 10C illustrates a closed configuration of the flow gate. The
flow gate can be closed by applying pressure 1077C (e.g., 20 PSIG) to the
thin flexible material adhered to the gating connector ports in the
biochip directly above the flow gate junction. By closing the connection
between the two channels of the flow gate, the fluid cannot flow from one
channel to another channel. In other embodiments, additional layers of
material are disposed above the gating ports to provide channels that
pass above the gate junction. Pressure applied to the channels passing
above the gate junctions distort the layers of material, thereby closing
the flow gate. The fluid gating connectors on the biochip can be
controlled by pneumatic pressure between slightly above 0 PSIG and 50
PSIG, but other pressure ranges are within the scope of the invention. In
another illustrative implementation, the flow gates are normally closed
and are opened by deforming material adjacent to the flow gate in order
to open the gate junction.

[0115] In another embodiment, pressure applied to the flexible membrane is
adjusted to control the flow rate of the fluid. Thus the flexible
membrane can be adjusted to a partially open configuration.

[0116] Some examples of materials for the flow-gate membrane include PTFE,
Teflon, PVC, polyethylene, polypropylene, polyimide, latex, silicone, and
nitrile. The thickness can, for example, range from ˜5 μm to
˜2 mm. The adhesive on the bottom side can be, for example, acrylic
adhesive and silicone adhesive. These examples are two common adhesives
on the market, but the scope of the invention covers other types of
adhesives. In some embodiments, the adhesive are applied to the membrane
prior to assembly onto the biochip by a spray method, film deposition
method, laminating method, etc. The thickness of the adhesive is
optionally kept as thin as possible and ranges between 5 um to 1 mm, for
example. In some embodiments, a thin adhesive layer and thin (flexible)
membrane configuration is preferred.

[0117] The scope of the invention is not restricted to the flow gate as
described above. Other types of flow gates that controls the fluid flow
between two channels are within the scope of the invention.

[0118] In one embodiment, the biochip is mated with an interface and
control instrument such that the pneumatic controls are disposed atop the
flow gate junctions. Each pneumatic control is individually addressable
to control flow fluid across a channel on either side of the flow gate.
In one embodiment, the interface and control instrument is configured to
open the various flow gates according to a programmed sequence at
predetermined times. In another embodiment, an individual using the
instruction actuated each flow gate manually. The interface and control
instrument can also be equipped with the heaters for sample preparation
and PCR or isothermal amplification, reagent and sample input mechanisms,
and fluorescent detection devices.

[0119] In some implementations, the interface and control instrument also
controls the introduction and flow of fluids in the biochip. For example,
one embodiment of the control instrument actuates (e.g., via a
servo-mechanical device) syringes, filled with a predetermined quantity
of liquid, that are attached to the LUER-LOK® connections at the
sample input port 755, the sample preparation wash buffer input port 760,
and the wash buffer input port 725. Alternatively, a cartridge with
pre-filled buffers can be assembled or affixed to the biochip such that
the input and output ports in the biochip are mated to the designated
prefilled buffers in the cartridge. The buffers can then be pneumatically
driven from the cartridge into the designated ports in the biochip.
Meanwhile, the elution buffer input port 765 and post-amplification
buffer input port 780 are supplied with their corresponding fluids by
pneumatic pressure applied to a quantity of fluid. In an illustrative
embodiment, pressures under 10 PSIG are used to supply the fluid to ports
765 and 780 (e.g., 0.5 PSIG). However, other pressures are within the
scope of the invention.

[0120] Any of the steps for using the biochips can be performed as part of
an automated process. For example, in one process for using the biochip
in a semi-automated fashion, an operator connects a prefilled buffer
cartridge or the various syringes and/or LUER-LOK® adapters to the
various ports on the biochip. The operator then places the biochip into a
chamber that is part of the interface and control instrument. The chamber
is designed such that individually addressable pneumatic actuators are
disposed above the various flow gates of the biochip. The control
instrument then conducts each step of the process up to the point of
detecting the fluorescence emitted by the separation and detection
chambers. At this point, the operator moves the biochip to another
chamber of the interface and control instrument that has one or more
optical readers to detect the fluorescence signal. One of skill in the
art will understand that this semi-automated process is merely
illustrative, and that all steps of the process can be performed in a
single chamber, which incorporates all controls, fluid interface
connections, and optical readers.

[0121] Referring again to FIG. 7, the features of an embodiment of biochip
700 have the following illustrative dimension. In one implementation, the
overall biochip size is about 9.9 cm (3.9 inches) by about 7 cm (2.75
inches). The channels 735 that carry the sample matrix and lysis buffer
also contains nano and microbeads (<10 μm), cell debris, and/or
other sample related material (e.g., plasma, proteins, and/or debris from
swabs used during the sample input). Such channels include, for example,
the channel from the sample input port 755 to the sample preparation
chamber 770 and the channel leading to the waste output port 730. Hence,
the dimensions of these channels are about 800 μm by 800 μm. This
relatively large channel cross section reduces the likelihood of clogging
of the channel when micro-beads or debris aggregate. In other
implementations, channel dimensions can be 300 μm to 2000 μm to
reduce channel clogging. Larger cross-sectional areas are also within the
scope of the invention. Channels which are expected to be generally free
from debris, magnetic beads, and/or other particles are about 500 μm
by 500 μm. However, larger or smaller dimensions are within the scope
of the invention (e.g., 50 μm to 2000 μm and beyond). In the
illustrative embodiment, all LUER-LOK® ports are standard size, which
enable the ports to accommodate reservoirs, syringes, and/or other Luer
taper interface connections that have volume storage capacity from, e.g.,
50 μL to 50 mL.

[0122] The separation and detection chambers 705 are about 3.5 mm in
diameter with a volume of about 40 μL. Such a size will accommodate an
optical sensor above the chamber that has an active capture area of 3 mm
diameter. The active capture area of the sensor will determine the size
of the detection chamber. Thus, other diameters for the separation and
detection chambers are envisioned for larger or smaller optical sensors.
Providing a chamber that is about 0.5 mm larger in diameter than the
active capture area of the sensor provides alignment tolerance between
the biochip and the optical sensor.

[0123] In the illustrative embodiment of biochip 700, the volume of the
amplification chamber 775 is 20 μL. In other implementations, the
amplification chamber can be as small as 10 nL to 50 μL. Meanwhile,
the volume of the sample preparation chamber 770 is about 20 μL and
has a width of about 1300 μm. In this embodiment, the volume of the
sample preparation chamber 770 is about the same size of the
amplification chamber 775 so that when the nucleic acids are eluted in
the sample preparation chamber 770, that volume can be directly passed
into the amplification chamber 775.

[0124] The post-amplification vent chamber 785 is about 40 μL. In some
implementations, the post-amplification vent chamber 785 volume matches
that of the separation and detection chambers 705. Thus, upon completion
of amplification, the sample volume moves to the post amplification vent
chamber 785 and the addition volume accommodates the post amplification
buffer solution, which is mixed with the amplification products inside
chamber 785. The total volume of post-amplification solution then
directly flows into the separation and detection chambers 705.

[0125] In an embodiment of the biochip 700 that includes a parallel flow
path for the separation and detection chambers 705, the total volume of
the post-amplification vent chamber 785 will be roughly equal to or
slightly larger than the volume of all of the separation and detection
chambers 705 combined. Thus, the post-amplification vent chamber 785 will
accommodate the amplified products as well as the needed amount of post
amplified buffer to supply a sufficient amount of post-amplification
solution to each of the separation and detection chambers 705.

[0126] As set forth above, the various vent chambers in biochip 700 enable
gas in the channels and chambers upstream of the corresponding vent
chamber to be removed from the biochip. Each vent chamber has a membrane
affixed to the top and/or bottom of the chamber. The membrane permits the
expulsion of gas while retaining liquid during liquid flow in the
biochip. The pore size of the membrane is about 0.45 μm. However, the
pore size can range from 0.01 μm to 2 μm. Non-limiting examples of
materials suitable for use as a vent membrane include Millipore 0.22
μm PTFE membrane material and Millipore SUREVENT® 0.45 μm PVDF
membrane material. The vent membranes are bonded to the biochip after
bonding the thin film to the substrate and creating holes in the thin
film corresponding to the vent chambers. The membranes can be affixed
using any number of adhesives known in the art, or the membranes can be
joined by thermal welding and/or ultrasonic welding. Membranes attached
in this way can withstand an internal pressure of at least 45 PSI. Thus,
this permits fluids to be loaded into channels and chambers up to a vent
membrane by keeping the fluid loading pressures below this amount (e.g.,
<10 PSI).

[0127] FIGS. 11A and 11B illustrate some embodiments of an integrated
biochip system, where an integrated biochip 1100 is connected to an
integrated cartridge 1195. FIG. 11A shows an angled view, and FIG. 11B
illustrates a bottom view of the integrated biochip 1100. The integrated
cartridge contains various reservoirs and provide buffer fluids to their
corresponding input ports.

[0128] FIG. 12 illustrates the integrated biochip without the cartridge.
The biochip 1200 uses a parallel detection approach, similar to the
biochip 500, and includes 8 detection chambers 1263. In some embodiments,
the separation and detection chambers are optionally made to be wide
meander channels rather than a circular chamber. Typically, if a circular
chamber is several times larger than the size of an incoming channel
(e.g. >2 or 3 times), then fluid will break apart when it flows from
the small channel to the large circular chamber. To reduce the improper
fillig of fluid, we introduce "a transition channel" as described above.
Using the transition channel, the size of the incoming channel gradually
increases to finally lead into the circular chamber. This method avoids
fluid breaking and bubbles in the chamber when the fluid flows at a slow
rate. However, when the fluid moves at a higher flow-rate, the fluid can
occasionally break-up and create air bubbles in the circular chamber even
with the transition channel. Thus, the circular chamber may not be
completely filled with the fluid. In contrast, a wide meander channels
can be more effective for smaller volumes (e.g. less than 20 uL).

[0129] In some embodiments, the meander channels (i.e. the separation and
detection chambers) are designed within a circular periphery. This design
enables a detection instrument to read the fluorescence signal for either
the `meander design` or `circular chamber design`. Thus, in some
embodiments the wide meander channel is replaced with a circular chamber.

[0130] Similar to the biochip 700, in some embodiments the biochip 1200
includes a sample preparation chamber 1270, an elution vent chamber
1245A, an amplification vent chamber 1245B, an amplification chamber
1275, a post-amplification vent chamber 1285, and flow gates 1240. The
biochip 1200 also optionally includes pneumatic/air lines 1233. Other
embodiments of the biochip includes some, but not all, of the components
listed above.

[0131] The elution vent chamber 1245A allows the air in the elution buffer
chamber to be vented outside of the biochip, and the amplification vent
chamber 1245B allows the air in the amplification chamber to be vented
outside of the biochip.

[0132] In some embodiments, the pneumatic/air lines 1233 provides
connection to the biochip assembly. As shown in FIG. 12, the system
interfaces all of the flow-connectors with pneumatic air-lines. Then, the
system can use the same interface to deliver air to the cartridge, rather
than a separate mechanism on top of the cartridge to supply air as
described above. In some embodiments, the pneumatic air lines deliver the
air from the biochip to the bottom of the cartridge. Then, the air is
routed through a vertical channel from the bottom to the top of the
cartridge and is delivered to the top of the fluid reservoirs. Each fluid
reservoir has a dedicated pneumatic air line from the biochip to the top
of the fluid reservoir in the cartridge.

[0134] In some embodiments, a pre-filled buffer cartridge is incorporated
as part of an assembled biochip. The buffers in the cartridge can be
driven pneumatically when the biochip is placed on the instrument and
interface with the pneumatic manifold in the instrument. The cartridge
also has optional empty chambers to serve as waste chambers for unloading
buffers after each bioassay process in the biochip. This cartridge
assembly can enable fully automated operation and eliminate the need for
the operator to use syringes and their connection to the biochip. The
assembly of the cartridge and biochip is illustrated below in connection
with the descriptions of FIGS. 15-17.

[0135] FIG. 13 illustrates the integrated cartridge 1195 separate from the
integrated biochip 1200. The integrated cartridge 1195 contains a number
of reservoirs connected to input ports on the integrated biochip 1200.
Exemplary reservoirs are waste reservoirs 1389 (connected to the waste
liquid output ports 1288), sample and lysis buffer reservoir 1356
(connected to the sample and lysis buffer input port 1255), an elution
buffer reservoir 1366 (connected to the elution buffer input port 1265),
a sample preparation wash buffer reservoir 1361 (connected to the sample
preparation wash buffer input port 1260), a post-amplification buffer
reservoir 1381 (connected to the post-amplification buffer input port
1280), and a separation wash buffer reservoir 1326 (connected to the
separation wash buffer input port 1225). In some embodiments, the
reservoirs is pre-filled with reagents. The waste liquid output
reservoirs 1389 are initially empty, but is filled up with the waste from
the separation and detection chambers. There can be multiple waste liquid
output reservoirs, or one large reservoir. In some embodiments, the
reservoirs have the same size, but the size can be different for
different reservoirs.

[0136] Some embodiments of the buffer cartridge 1495 have a rubber
material between the top cover and the cartridge body. In some
embodiments, there exists an opening on the top cover for the sample
introduction (for example from a syringe, pipette, or even to introduce a
swab top into the cartridge). The rubber directly below this opening has
a sealable opening. In some embodiments, the sealable opening is a slit.
When a pipette, syringe, or swab, is thrust into the rubber slit, the
rubber opens to provide access to the reservoir in the buffer cartridge
to enable the user to deposit the sample inside the cartridge. When the
pipette/syringe is retracted, the elasticity of the rubber ensures that
it closes the slit, shutting-off access to the reservoir. This air tight
self-seal created by the rubber ensures that any environmental
contaminant is not subsequently introduced into the reservoir and the
self-seal also prevents or minimizes pneumatic/air leak from the
reservoir when the buffer/sample is driven pneumatically from this
reservoir in the cartridge into the biochip.

[0137] FIGS. 14A and 14B illustrates embodiments of a buffer cartridge
1495 with a self-sealing rubber gasket. In some embodiments, the
cartridge 1495 includes sample reservoirs 1490 and a rubber material 1492
on top of the reservoirs. On top of the rubber material 1492, there is a
top cover 1493, which can be plastic. In some embodiments, the top
plastic, rubber, cartridge and biochip are affixed/bonded to constitute a
fully assembled biochip with the integrated buffer cartridge 1495. The
top cover has a hole 1462, to which a pipette 1467, syringe or swab can
be introduced. Immediately below the hole 1462, there is a rubber slit
1468 that can be open to provide access to sample reservoirs 1490. In
some embodiments, the slit is a straight line, a `X` pattern, or a `star`
pattern.

[0138] In some embodiments, the thickness of the rubber material 1492
ranges from 0.5 mm to 50 mm. The material is, for example, silicone,
PTFE, neoprene, polyethelene, polymide, polycarbonate, acrylic,
cyclo-olyfin polymers, cyclo olefin polymers, or any other material that
has elasticity similar to that of rubber or elastomer. The durometer for
example is Shore A 50 durometer, as used in one demonstration. The slit
1468 in the rubber that provides access to the sample reservoir below is,
for example, between 0.5 mm and 25 mm, or larger as required. Thicker
rubber and/or harder material durometer will require higher mechanical
force by the operator to open the slit in the rubber to access the sample
reservoir in the cartridge. Nevertheless, thicker rubber and/or harder
material durometer can be advantageous as they can provide better
self-sealing and can prevent or reduce air leak when pneumatic pressure
is applied inside the sample reservoir in the cartridge to drive the
sample into the biochip.

[0139] In another exemplary configuration, the hole 1462 has a diameter of
˜4 mm, and the top cover 1493 is ˜2 mm thick. Also, the slit
size is ˜2.5 mm, and the top cover is an acrylic cover. The rubber
material 1492 beneath the top cover 1493 is ˜3 mm thick and Shore
A, 50 durometer.

[0140] FIG. 15A illustrates a cartridge, membrane and biochip stack bonded
together according to some embodiments. In some embodiments, the biochip,
membrane, and cartridge are manufactured separately as independent
modules and then be mated and joined/bonded. In alternative embodiments,
the biochip and cartridge are fabricated as a single unit. The stack can
be bonded by adhesive or clamped by various mechanisms. The cartridge
1595 has a hole 1563 to allow fluid to flow out of the cartridge to the
biochip 1500. The membrane 1511 has a slit 1513 that controls the flow of
fluid 1503 from the cartridge 1595 to the biochip 1500. In some
embodiments, the slit 1513 is located anywhere above the chamber 1517 of
the biochip. To provide area for the slit 1513 to open, the chamber 1517
is optionally made larger than input ports of biochip 700. In FIG. 15, by
placing the slit 1513 offset from the hole 1563 (i.e., not directly under
the hole), leakage through the slit is prevented or minimized when the
slit is not open and/or there is backpressure from the fluid/air from the
biochip to the reservoir. If the slit was located directly under the
hole, there is no object blocking the slit from opening upward when there
is backpressure (i.e. fluid trying to enter from the biochip into the
cartridge-reservoir). The membrane will stretch upwards into the hole and
eventually open as the fluid pressure from the biochip increases allowing
fluid flow in the reverse direction. However, if the slit 1513 is offset
from the hole 1563, the slit cannot open because it will be thrust
against the body of the cartridge.

[0141] Other properties of the membrane and the biochip are described
below. Some of the exemplary materials of the membrane are silicone,
latex, PTFE, and polyethylene. In some embodiments, thickness ranges from
0.001'' to 0.1''. The thicker the material, the more pressure required to
open the slit 1513 and to enable fluid flow across membrane through the
slit. Some embodiments of the chamber 1517 are larger than the size of
the hole 1563 of the cartridge. The chamber is connected to a channel
1501 through which the fluid will flow to reach its destination.

[0142] FIG. 15B is a B-B cross-sectional view of the cartridge 1595. FIG.
15C is a C-C cross-sectional view of the membrane 1511 with the slit
1513. FIGS. 15D and 15E are DE-DE cross-sectional views of the biochip
1500. Different embodiments of the chamber 1517 of the biochip have
different shapes (e.g., slot, circular, oval).

[0143] FIG. 16A illustrates a cross-sectional side view of the stack of
FIG. 15A with an open membrane slit according to an embodiment of the
invention. When pneumatic/air pressure 1677 is applied from the top of
the cartridge 1595, the pressure will be applied downward to the slit
1513 and open the slit. Then the fluid flows from the cartridge 1595 to
the biochip 1500. FIG. 16B is a B-B cross-sectional view of the membrane
1511 with the open slit 1513.

[0144] FIG. 17A illustrates a cross-sectional side view of the stack of
FIG. 15A with a closed membrane slit according to an embodiment of the
invention. Unlike the downward pressure 1677 of the FIG. 16A, the
pressure 1777 from the other side of the channel will create upward
pressure on the membrane 1511. As illustrated above, because the bottom
portion of the cartridge 1595 is placed against the membrane 1511, the
bottom portion of the cartridge prevents the slit 1513 from opening
upward. As a result of the closed slit, fluid cannot flow from biochip to
the cartridge. FIG. 17B is a B-B cross-sectional view of the membrane
1511 with the closed slit 1513.

[0145] In the examples provided above, only one type of DNA probe was
included in each of the separation and detection chambers and one type of
fluorescent label was used. However, because the separation and detection
chambers physically separate the different DNA fragments via binding to
the DNA probes, multiple types of DNA probes and multiple types of
fluorescent dyes can be used in other implementations. For example, a
biochip that is able to separate and detect 30 different types of DNA
fragments in six different separation and detection chambers is within
the scope of the invention. In such an embodiment, a collection of 30
different types of amplification primers (either by PCR or other
amplification methods) are supplied that include only five different
fluorescent labels, each label having a different emission color. Thus,
after amplification (assuming each DNA target fragment is present in the
sample), the post-amplification solution will contain a collection of 30
different types of DNA fragments, but these fragments will only be
labeled with five distinct colors. Using known methods, one would not be
able to distinguish the DNA fragments that are labeled with the same
color from each other.

[0146] In embodiments of the present invention, five different DNA probes
can be immobilized in each of the six separation and detection chambers,
for a total of 30 different types of DNA probes in the biochip as a
whole. Each of the five different types of DNA probes in a particular
chamber will correspond to a different label color. In this way, the
biochip captures five different DNA fragment types per chamber, each
being labeled with a different color fluorescent label. Thus, because
each chamber now only has one type of DNA fragment per color in each
chamber, the interference between the labels emitting the same color is
reduced or eliminated.

[0147] Implementations of the invention enable a gas to be introduced into
one or more of the separation and detection chambers after the target DNA
fragments have been bound to the DNA probes immobilized therein. This
enables the removal of substantial amounts of any buffers or other
non-bound material from the separation and detection chamber, thereby
enabling the fluorescence to be detected on a dry basis.

[0148] One having ordinary skill in the art will recognize that the
embodiments set forth herein are merely illustrative of the present
invention. Thus, the techniques, devices, and system described above can
be modified to enable known genetic material amplification methods to be
performed while remaining within the scope of the invention.

Testing Platform

[0149] FIG. 18 depicts a genetic testing platform 1800 including a compact
processor unit 1810 and a table-top reader unit 1820 according to
embodiments of the present invention. In some embodiments, the units draw
power from a standard 110V/220V wall outlet. The processor unit includes
a bottom rapid peltier based thermocycler module to provide heat required
for cell lysis, DNA elution, reverse-transcriptase and amplification. The
processor also includes a top programmable pneumatic (0-100 psig) module
1830 for fluid flow control in the integrated biochip 1801. In
alternative embodiments, there are two or more separate units for
providing heat, providing pressure, and reading fluorescence.

[0150] In some embodiments, the reader unit is FDA classified for in-vitro
diagnostic use. The 2-unit instrument platform offers significant capital
cost savings for customers who desire to simultaneously run several
samples on individual biochips (e.g., on-demand, Random Access Testing).
For example, customers can easily set up a bench-top work station with a
number of processor units (e.g. 2 to 50) and only one reader unit.
Multiple samples can be simultaneously prepared on the biochip in less
than 5 minutes per sample, tested in individual processor units in about
90 minutes, and sequentially read by the reader unit in less than 2
minutes per chip.

[0151] In some embodiments, to perform a diagnostic test, the integrated
biochip is placed in the processor unit between the top pneumatic module
and bottom peltier module. This stand-alone setup enables the integrated
biochip to perform diagnostic test processes such as cell lysis, nucleic
acid extraction, nucleic acid capture, nucleic acid purification, reagent
mixing, reverse-transcriptase reaction and amplification, sequentially
executed in an integrated manner in the disposable biochip. Typical
processing time from providing sample to complete multiplex amplification
is under 90 minutes. Upon completion, the integrated biochip is
transferred to the table-top reader unit for a fluorescence read of
amplified DNA fragments, which is typically accomplished in under 2
minutes. The reader unit has a multi-color excitation-emission optical
filter system and utilizes highly sensitive photomultipler tube
detection. This reader demonstrated to accurately detect fluorescent
signal from even a single copy of amplified DNA.

Experimental Data

[0152] This section provides test data for the biochip and method
described above. Ultramer/DNA of 4 respiratory pathogens (purchased from
IDT Technologies, CA), namely, Influenza-A, Influenza-B, Influenza-A/H1
and Influenza-A/H3, at 100000 copies each were spiked into 10 μL of
human blood sample. FIG. 19A lists the forward primer, reverse primer,
probe sequences of the four respiratory pathogens mentioned above. FIG.
19B lists two common quencher sequences that are used in this experiment.
The sample was then used for processing on biochip.

[0153] A common quencher oligonucleotide is a random DNA sequence of 8-20
bp lengths. A common quencher oligonucleotide with a dye-quencher moiety
can be used for quenching the fluorescence of the probe. For example, in
the first sequence of FIG. 19B, the random DNA sequence is 5'-TGTTATTCAGT
and the dye-quencher moiety is 3IAbRQSp. The probe includes a gene
specific sequence to bind with a target DNA and a common quencher
complementary sequence (e.g., complementary sequence to TGTTATTCAGT) to
bind with a common quencher. The common quencher complementary sequence
of the probe contains fluorescence, and when a common quencher
oligonucleotide binds to the probe, the fluorescence of the probe will be
quenched. This configuration of probe allows binding of a common quencher
oligonucleotide to the probe containing a complementary quencher
sequence. Thus, one or few common quencher oligonucleotides can be used
for a plurality of probes designed to bind to their corresponding DNA
targets. The detailed description of the probe and the common quencher
oligonucleotide can be found in the incorporated application, entitled
"Method for Separation and Detection of DNA Fragments."

[0154] The sample prep-process utilized the reagents from a commercially
available kit, easyMAG® (bioMerieux, Inc, NC) which includes the lysis
buffer, magnetic beads for nucleic acid capture, wash buffer and elution
buffer. Briefly, the spiked sample was input into 100 μL of lysis
buffer containing magnetic beads to capture nucleic acid. The solution
was incubated for about 5 min.

[0155] After incubation, pneumatic pressure provides the flow of such
solution through the sample preparation chamber capturing the magnetic
beads inside the chamber since a magnet was positioned above (and
outside) the sample-prep chamber. The retained magnetic beads would have
nucleic acid bound to it from earlier the lysis incubation chemistry.
Next, wash buffer 1000 μL that flowed through the magnetic beads
washed the beads and removed components of the lysis buffer (for example,
Chaotropic salts) and other biological elements (e.g. extracellular
compounds, proteins). Subsequently, the introduction of the elution
buffer 20 μL to the magnetic beads and incubation for about 3 min
released the nucleic acid from the magnetic beads into the elution
buffer. Next, pneumatic pressure moved the elution buffer containing the
nucleic acid/DNA into the PCR/isothermal amplification chamber. The
elution bufferwas mixed with Qiagen multiplex PCR Kit (cat#206152,
Qiagen, CA), HotStar polymerase (1U per reaction) and forward and reverse
primers for each of the 4 DNA targets at concentration of 0.2 μM each.
The designed primers for the 4 targets are listed in FIG. 19 below.
Multiplex PCR was then performed in which all for targets are amplified
in the same reaction. PCR thermal cycling conditions were, 96° C.
for 10 min for initial denaturation followed by 40 cycles of 96°
C. for 1 min, 60° C. for 1 min, 72° C. for 1 min, and final
extension at 72° C. for 5 min.

[0156] Upon completion of PCR amplification, the PCR amplicons moved to
the post-amplification vent chamber where it was mixed with about
˜70 μL of the post-amplification buffer, in this case deionized
water, to a volume of ˜90 μL including the PCR amplicons from
the PCR chamber. Next, pneumatic pressure applied to the input channels
moved the solution from the `post-amplification vent chamber` into 4
detection chambers. Each of the four detection chambers contained probe
and quencher for only one targets, all labeled with FAM dye. When the 90
μL of reconstituted PCR amplicons flowed into the four detection
chambers, it was mixed with the probe and quencher present in its
respective chambers. Chamber 1 contained the probe and quencher for
Influenza-A, chamber 2 for Influenza-B, chamber 3 for Influenza-A/H1 and
chamber 4 Influenza-A/H3. The probe and common quencher sequence for
these targets is also listed in FIG. 19. The probe and the
common-quencher oligo containing BHQ quencher were at equimolar
concentration of 0.2 μM contained in ˜5 μL solution and all
probes were labeled with Cy5 dye. With ˜25 μL capacity in each
detection chamber, approximately 20 μL of the reconstituted PCR
amplicon was filled into each chamber simultaneously. Closing the flow
gates leading into an unused detection chamber restricted the flowinto
unused detection chambers.

[0157] Once the PCR amplicon was loaded into all 4 detection chambers
simultaneously, the temperature was ramped from ˜25° C.
(room temperature) to 94° C. at the rate of 1.5°
C.-2.5° C. per second. After the temperature reached 94°
C., from the temperature was ramped down from 94° C. to 25°
C. at a ramp down rate of 2° C.-4° C. per second. This ramp
up and ramp down allowed for the different denaturing and annealing
interactions between the PCR amplified amplicons, probes and primers, as
described in the incorporated application: "Method for Separation and
Detection of DNA Fragments." Finally, a fluorescence reader (FLX800T,
BioTek Instruments, VT) as shown below measures the fluorescence of Cy5
in each of the 4 chambers. Presence of Cy5 fluorescence in each chamber
indicated the successful binding of a PCR amplicon to the corresponding
detection probe present in that chamber. FIG. 20 shows the increases in
probe fluorescence [in relative fluorescence units (RFU)] for each of the
4 targets (Influenza-A, Influenza-B, Influenza-A/H1, Influenza-A/H3).
Using this detection method of the present invention and using only
single color fluorescence, we demonstrated the detection of 4 targets in
this example.

[0158] As will be apparent to one of ordinary skill in the art from a
reading of this disclosure, the present disclosure can be embodied in
forms other than those specifically disclosed above. The particular
embodiments described above are, therefore, to be considered as
illustrative and not restrictive. Those skilled in the art will
recognize, or be able to ascertain, using no more than routine
experimentation, numerous equivalents to the specific embodiments
described herein. The scope of the invention is as set forth in the
appended claims and equivalents thereof, rather than being limited to the
examples contained in the foregoing description.